Phenotypic divergence in widespread plants : genetic drift, selection and plasticity

This thesis presents studies that describe and explain phenotypic differentiation within several alpine plant species. The key elements that are addressed are threefold: (1) effects of neutral genetic drift, natural selection and phenotypic plasticity on phenotypic differentiation; (2) effects of glacial history, geography and climate on phenotypic differentiation and adaptation; (3) genetic structure and gene flow at small spatial scale. Combining all three elements, the aim of this thesis is to understand how a plant species' evolution towards its current state is affected at different spatial scales by neutral genetic drift and historical (i.e. glaciation-related) as well as more recent (i.e. postglacial) environmental influences. To measure phenotypic differentiation in important plant traits, common garden experiments were performed with several alpine plant species (Campanula thyrsoides, C. barbata, Geum reptans) sampled from populations across the European Alps and Jura Mountains. Phenotypic differentiation was generally mirrored by molecular differentiation into distinct phylogeographic groups, which is explained by long-term survival in isolated glacial refugia. The results therefore suggest that glacial history affected not only the species' neutral genetic structure but also its phenotype. For some traits and in some regions, such differentiation could be explained as adaptation to the regional environment. For instance, the distinct phenology in Campanula thyrsoides, showing delayed flowering in the submediterranean southeastern Alps contrasting with early flowering at higher elevation in the other regions to the west, is clearly an adaptation to season length in the respective environments. Differentiation in various other traits could not be explained as adaptations and may therefore be due to drift alone. Postglacial adaptation was detected when correlating trait values with altitude of origin. For instance, the negative correlation of altitude with plant height in Campanula thyrsoides, achieved without compromising flower production, is probably an adaptation to harsher conditions and to increased investment in roots. Adaptation can also occur through phenotypic plasticity. In an experiment in which Campanula thyrsoides was grown in common gardens at three different altitudes, variability in the functional trait of specific leaf area could be dissected into a constitutive genetic part and a phenotypic plastic part. At the local scale, populations of C. thyrsoides were considerably differentiated in neutral molecular markers, which could be due to founder effects in the recent past. Experimental studies showed that seed dispersal was also limited in the landscape. Within-population genetic diversity was found to be high and probably the result of strong self-incompatibility and outcrossing in this species. In line with this, a paternity analysis showed that pollen dispersal is well-mixed within the investigated population, but a substantial amount of pollen was derived from neighbouring populations in this specific landscape, indicating ongoing mixture. To conclude, the studies described in this thesis showed that glacial history had strong effects on phenotypic differentiation, and that part of this differentiation is due to adaptation to past as well as current conditions, whether through constitutive genetic adaptation or phenotypic plasticity - though neutral genetic drift may also have a substantial contribution to differentiation. At the local scale, the heterogeneity of the European Alps and the particular autecology of alpine species may have contributed to local differentiation

Forest wildflowers bloom earlier as Europe warms: lessons from herbaria and spatial modelling

Today plants often flower earlier due to climate warming. Herbarium specimens are excellent witnesses of such long-term changes. However, the magnitude of phenological shifts may vary geographically, and the data are often clustered. Therefore, large-scale analyses of herbarium data are prone to pseudoreplication and geographical biases. We studied over 6000 herbarium specimens of 20 spring-flowering forest understory herbs from Europe to understand how their phenology had changed during the last century. We estimated phenology trends with or without taking spatial autocorrelation into account. On average plants now flowered over 6 d earlier than at the beginning of the last century. These changes were strongly associated with warmer spring temperatures. Flowering time advanced 3.6 d per 1°C warming. Spatial modelling showed that, in some parts of Europe, plants flowered earlier or later than expected. Without accounting for this, the estimates of phenological shifts were biased and model fits were poor. Our study indicates that forest wildflowers in Europe strongly advanced their phenology in response to climate change. However, these phenological shifts differ geographically. This shows that it is crucial to combine the analysis of herbarium data with spatial modelling when testing for long-term phenology trends across large spatial scales

Sustainable seed harvesting in wild plant populations

Seed harvesting from wild plant populations is key for ecological restoration, but may threaten the persistence of source populations. Consequently, several countries have set guidelines limiting the proportions of harvestable seeds. Here, we use high-resolution data from 298 plant species to model the demographic consequences of seed harvesting. We find that the current guidelines only protect some species, but are insufficient or overly restrictive for others. We show that the maximum possible fraction of seed harvesting is strongly associated with harvesting frequency and generation time of the target species, ranging from 100% in long-lived species to <1% in the most annuals. Our results provide quantitative basis to guide seed harvesting legislation based on species’ generation time and harvesting regime

Recent evolution of flowering time across multiple European plant species correlates with changes in aridity - Greenhouse data

Ongoing global warming and increased drought frequencies have a large impact on plant populations and potentially drive evolutionary adaptations. Historical comparisons, where plants grown from seeds collected in the past (“ancestors”) are compared to plants grown from freshly collected seeds from the same populations (“descendants”), are a powerful method to investigate such evolutionary changes across many taxa. When applied to multiple species simultaneously, historical comparisons can reveal recent parallel evolutionary shifts. We used 21-38 year old seeds of 13 European plant species, stored in seed banks and originating from Mediterranean and temperate regions, for a greenhouse experiment that investigated shifts of flowering phenology, as a potential result of adaptive evolution to increased drought over the last decades. We additionally used single nucleotide polymorphism (SNP) markers to quantify relatedness and levels of genetic variation, and to characterize potential neutral processes and differences in sampling schemes. We found that, across species, descendants grew faster and advanced their flowering, and that these shifts were correlated with changes in aridity at the population origins, suggesting that drought induced evolution of earlier flowering. In 6 out of the 13 species, however, the SNP markers detected strong differences in genetic variation and relatedness between ancestors and descendants, indicating that other evolutionary processes may have contributed to genetic changes. Our results suggest that changes in aridity due climate change may have influenced the evolutionary trajectories of many plant species in different regions of Europe, and that flowering phenology may be one of the key traits that is rapidly evolving. Our study provides further evidence that seed bank collections, with some limitations, are a largely untapped resource for investigating the impact of global environmental changes on plant populations

Monitoring rapid evolution of plant populations at scale with pool-sequencing

The change in allele frequencies within a population over time represents a fundamental process of evolution. By monitoring allele frequencies, we can analyze the effects of natural selection and genetic drift on populations. To efficiently track time-resolved genetic change, large experimental or wild populations can be sequenced as pools of individuals sampled over time using high-throughput genome sequencing (called the Evolve & Resequence approach, E&R). Here, we present a set of experiments using hundreds of natural genotypes of the model plant Arabidopsis thaliana to showcase the power of this approach to study rapid evolution at large scale. First, we validate that sequencing DNA directly extracted from pools of flowers from multiple plants -- organs that are relatively consistent in size and easy to sample -- produces comparable results to other, more expensive state-of-the-art approaches such as sampling and sequencing of individual leaves. Sequencing pools of flowers from 25-50 individuals at ∼40X coverage recovers genome-wide frequencies in diverse populations with accuracy r > 0.95. Secondly, to enable analyses of evolutionary adaptation using E&R approaches of plants in highly replicated environments, we provide open source tools that streamline sequencing data curation and calculate various population genetic statistics two orders of magnitude faster than current software. To directly demonstrate the usefulness of our method, we conducted a two-year outdoor evolution experiment with A. thaliana to show signals of rapid evolution in multiple genomic regions. We demonstrate how these laboratory and computational Pool-seq-based methods can be scaled to study hundreds of populations across many climates